Ramiro Muniz-Diaz thanks CONACyT for the scholarship.

1. Bacterial isolation and identification
<ol>
	<li>Isolation of an endophytic strain from garlic bulb meristems:
	<ol>
		<li>Place 2 mm fragments of meristems from previously peeled, disinfected, and cut garlic bulbs on trypticase soy agar (TSA) as a rich growth medium, and incubate at 25 &#176;C for 1 day.</li>
		<li>Based on the morphological differences of the bacterial colonies-shape, size, color, edge, transmitted light, reflected light, texture, consistency, and pigment production-describe the different observed morphotypes, and purify by cross-streaking a representative colony of each morphotype.</li>
		<li>Preserve all the purified bacterial strains in 30% glycerol at &#8722;80 &#176;C.</li>
		<li>Identify a randomly selected strain (9AP) by sequencing the 16S rDNA. Extract the DNA following the method of Hoffman and Winston8.</li>
		<li>Amplify the 16S rRNA by polymerase chain reaction (PCR) with 100 ng of DNA, 1x polymerase buffer, 4 mM MgCl2, 0.4 mM dNTPs, 10 pmol each of 27F/1492r universal oligonucleotides, and 1 U of DNA polymerase9. Use the following thermal cycler conditions: 95 &#176;C for 5 min for the initial denaturalization; followed by 35 cycles of 1 min at 94 &#176;C as the denaturalization temperature, 1 min at 56 &#176;C as the hybridization temperature, and 1 min at 72 &#176;C as the extension temperature; and then 10 min at 72 &#176;C as a final extension.</li>
		<li>Capillary-sequence the PCR product using the same oligonucleotides; use the dideoxy-terminal method with the matrix installation kit for capillary sequencing10, and perform electrophoresis in an automated multicapillary system11.</li>
		<li>Manually edit the sequences, compare the sequence with the GenBank library using a BLAST search, and tentatively identify the strain with the gold standard of at least 98.7% identity with the closest species12,13. 
		NOTE: The strain 9AP was identified as belonging to the species of Pseudomonas hunanensis, with an identity of 99.86% with the sequence for the strain P. hunanensis LV (JX545210).</li>
	</ol>
	</li>
</ol>
2. Bacterial sample preparation for morphological observation by AFM
<ol>
	<li>Under sterile conditions, place a drop of the bacterial suspension (Pseudomonas hunanensis, Staphylococcus aureus) on a glass slide.</li>
	<li>Fix the samples on the slide by dry heating. Pass the slide gently over a flame several times until the sample is dry. 
	NOTE: The fixation of samples is a routine technique in microbiology to observe fixed prokaryotic organisms and to simulate their structure as living cells as closely as possible.</li>
	<li>Place the fixed samples in a Petri dish, and take them to the AFM equipment for observation. 
	&#8203;NOTE: As the samples can be altered by weather conditions over time, set aside a maximum time of 24 h for analysis in the AFM equipment. Keep the samples free of dust.</li>
</ol>
3. Antibacterial effect of MgO nanoparticles against bacteria
NOTE: The synthesis and characterization of MgO NPs have been published previously14. In this work, the antibacterial activity of the nanomaterials was estimated based on the Clinical and Laboratory Standards Institute (CLSI) manual using macrodilution and microdilution methods for inhibition15,16.
<ol>
	<li>Estimation of the minimum inhibitory activity (MIC) and bactericides (CMB)
	<ol>
		<li>Pregrowth of the strains
		<ol>
			<li>Inoculate an appropriate amount of Escherichia coli or Staphylococcus aureus into nutritious broth to obtain a final concentration of 1 &#215; 108 CFU/mL.</li>
			<li>Incubate the suspension at 37 &#176;C (&#177; 2 &#176;C) for 18-24 h.</li>
		</ol>
		</li>
		<li>Postgrowth of acclimatized strains
		<ol>
			<li>Immerse a sterile bacteriological loop in the suspension, touching the bottom of the tube.</li>
			<li>Perform streaking with the loop on eosin and methylene blue agar and nutrient agar.</li>
			<li>Incubate the agar plates at 37 &#176;C (&#177; 2 &#176;C) for 24 h.</li>
		</ol>
		</li>
		<li>Selection of colonies for the preparation of the standardized inoculum
		<ol>
			<li>Take three to five colonies with the same morphological type by touching the top of the colony in the Petri dish with a bacteriological loop.</li>
			<li>Transfer and immerse the selection in 3-5 mL of M&#252;ller-Hinton broth.</li>
			<li>Incubate at 37 &#176;C (&#177; 2 &#176;C) to achieve a turbidity of 1 &#215; 108 CFU/mL or 2 &#215; 108 CFU/mL. 
			NOTE: In this work, McFarland turbidity standards were used as a reference for the bacteriological suspensions; specifically, the 0.5 standard corresponds approximately to a homogeneous E. coli suspension of 1.5 &#215; 108 cells/mL. A UV-Vis spectrophotometer was used to measure the turbidity.</li>
			<li>Take 1 mL, and add it to 9 mL of sterile broth. Take 200 &#181;L of this dilution, and add it to 19.8 mL of sterile broth to obtain a final concentration of 5 &#215; 105 CFU/mL.</li>
		</ol>
		</li>
		<li>Required concentrations of MgO nanoparticles
		<ol>
			<li>Use an ultrasonicator for the preparation of the solutions.</li>
			<li>Suspend the nanomaterial in sterile water at twice the concentration required to obtain serial solutions.</li>
			<li>Add these suspensions to sterile tubes containing M&#252;ller-Hinton broth to achieve the new range of concentrations required for the experiment. 
			NOTE: The following MgO NPs concentrations were applied for microdilution: 30 ppm, 60 ppm, 120 ppm, 250 ppm, 500 ppm, 1,000 ppm, 2,000 ppm, 3,000 ppm, 4,000 ppm, and 8,000 ppm.</li>
		</ol>
		</li>
		<li>Preparation of the microdilution15
		<ol>
			<li>Prepare a new and sterile 96-well microtiter plate (microplate). Label column No. 12 of the microplate as a sterility column; label column No. 11 as a growth control column.</li>
			<li>Add 120 &#181;L of the nanoparticle suspensions with the different concentrations to columns No. 1-10.</li>
			<li>Inoculate 120 &#181;L of bacteria (5 &#215; 105 CFU/mL) into the wells in columns No.1-10. 
			NOTE: For this study, row G and row H were controls with ceftriaxone at concentrations suggested by CLSI standards: 8 ppm, 4 ppm, 2 ppm, 1 ppm, 0.5 ppm, 0.25 ppm, 0.125 ppm, 0.06 ppm, and 0.03 ppm.</li>
			<li>Incubate the 96-well microplate for 24 h at 37 &#176;C (35 &#176;C &#177; 2 &#176;C). Finally, analyze the wells.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Observation of the MgO nanoparticle-induced morpho-structural changes in the bacterial strains by AFM
	<ol>
		<li>Take 250 &#181;L from the wells of the microdilution tests, mix with 750 &#181;L of sterile water, and centrifuge at 2,000 &#215; g from 2 min to 5 min at 5 &#176;C.</li>
		<li>Wash the precipitates three times with 1 mL of sterile water each time, and at the end of the washes, add 500 &#181;L of water to them.</li>
		<li>To obtain the AFM images, take 10-20 &#181;L of the final suspension of each starting well, perform a smear on a slide previously cleaned by ultrasound (30 min), and dry at room temperature for 1 h.</li>
	</ol>
	</li>
</ol>
4. AFM measurements
NOTE: Here, the atomic force microscope in contact mode was mounted on an anti-vibration workstation that allowed the isolation of the microscope from any mechanical vibrational sources and kept the system leveled. Electrical interference is diminished with line filters and surge protection. The AFM used here auto-aligns the laser beam to the photodetector.
<ol>
	<li>Turn on the computer and the AFM, then select the Contact mode, the contAI-G probes, and the Auto Slope options in the software tools. The default values of the Z-controller are Setpoint = 20 nM, P-Gain = 10,000, I-Gain = 1,000, D-Gain = 0, and Tip voltage = 0 mV.</li>
	<li>Place the samples in the AFM contact mode using a 70 &#181;m scan range head. We used ContAI-G silicon cantilevers with a spring constant of 0.13 N/m.</li>
	<li>Use a camera device mounted on two lenses integrated into the scan head to obtain a quick view of the surface of the area under the probe.</li>
	<li>Manually perform a displacement in the XY plane in order to choose the desired area. The probe is made to electronically approach the surface sample until the contact conditions are reached. Electronically adjust the XY measurement plane of the scanner and sample surface by reducing the slopes in the XY directions.</li>
	<li>Use the standard parameters of the software: 1 line per second at 256 points per line.</li>
	<li>First, perform a full scan range of a 70 &#181;m x 70 &#181;m area; then, select a smaller area using the zoom tool. The AFM optimizes the chart range in Z automatically. 
	NOTE: By decreasing the lines per second time, the total scan time increases, the quality of the scan is more defined, and some noise from the measurement is also cleared.</li>
	<li>In order to select a new area from the sample, retract the cantilever electronically, and move the sample along the XY plane. Realize a new scan using the procedure described in step 4.5 and step 4.6. 
	NOTE: The scans obtained are shown in a single-color bar scale, in which the light colors are associated with higher altitudes, and the dark colors are associated with deeper altitudes. The AFM software generates a 3D view of the scan that allows the appreciation of the details of the measured surface.</li>
	<li>Perform the data processing using the line by line leveling in the Tools menu of the image in the filter selection, which is the most used and simplest leveling method. 
	NOTE: This leveling method takes every horizontal or vertical line produced in the AFM image and fits it to a polynomial equation.</li>
	<li>Optimize the maximum and minimum height in the color bar on the right in order to obtain the best image resolution.</li>
	<li>Perform the image analysis by using the Analysis menu in the tools bar. Determine the heights and distances by selecting the initial and final points once the desired tool is selected. 
	NOTE: Users must try the different filters of the Tools menu to avoid tip-image artifacts due to the flattening used in the leveling process. Image artifacts appear as streak lines and are shown in some of the AFM images.</li>
</ol>

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The authors declare that they have no conflicts of interest.

Microscopy is a technique commonly used in biological laboratories that allows for the investigation of the structure, size, morphology, and cellular arrangement of biological samples. To improve this technique, several types of microscopes can be used that differ from each other in terms of their optical or electronic characteristics, which determine the resolution power of the instrument.
In scientific research, the use of microscopy is required for the characterization of bacterial cells; f...

Different bacterial shapes were first noted by Antony van Leeuwenhoek in the 17th century1. Bacteria have existed in a great diversity of shapes since ancient times, ranging from spheres to branching cells2. Cell shape is a fundamental condition for bacterial taxonomists to describe and classify each bacterial species, mainly for the morphological separation of gram-positive and gram-negative phyla3. Several elements are known to determine bacterial cell forms, all of which are involved in the cell covers and support as components of the cell wall and membrane, as well as in the cytoskeleton. In this way, scientists are still elucidating the chemical, biochemical, and physical mechanisms and processes implicated in determining bacterial cell forms, all of which are defined by clusters of genes that define bacterial shapes2,4.
Additionally, scientists have shown that the rod shape is likely the ancestral form of bacterial cells, since this cell shape appears optimal in cell-significant parameters. Thus, cocci, spiral, vibrio, filamentous, and other forms are regarded as adaptations to various environments; indeed, particular morphologies have evolved independently multiple times, suggesting that the shapes of bacteria could be adaptations to particular environments3,5. However, throughout the bacterial cell life cycle, the cell shape changes, and this also occurs as a genetic response to damaging environmental conditions3. The bacterial cell shape and size strongly determine the stiffness, robustness, and surface-to-volume ratio of the bacteria, and this characteristic can be exploited for biotechnological processes6.
Electronic microscopy is used to study biological samples due to the high magnification that can be reached beyond light-based microscopes. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) are the most commonly used techniques for this purpose; however, samples require some treatments before they are placed into the chamber of the microscope in order to obtain appropriate images. A gold cover on the samples is required, and the time used for total image acquisition should not be too long. In contrast, atomic force microscopy (AFM) is a technique widely used in the analysis of surfaces but is also employed in the study of biological samples.
There are several types of AFM modes used in surface analysis, such as contact mode, non-contact mode or tapping, magnetic force microscopy (MFM), conductive AFM, piezoelectric force microscopy (PFM), peak force tapping (PFT), contact resonance, and force volume. Each mode is used in the analysis of materials and provides different information about the surface of the materials and their mechanical and physical properties. However, some AFM modes are used for the analysis of biological samples in vitro, such as PFT, because PFT allows for obtaining topographical and mechanical data on cells in a liquid medium7.
In this work, we used the most basic mode included in every old and simple AFM model-the contact mode. AFM uses a sharp probe (around &#60;50 nm in diameter) to scan areas less than 100 &#181;m. The probe is aligned to the sample in order to interact with the force fields associated with the sample. The surface is scanned with the probe to keep the force constant. Then, an image of the surface is generated by monitoring the motion of the cantilever as it moves across the surface. The gathered information provides the nano-mechanical properties of the surface, such as the adhesion, elasticity, viscosity, and shear.
In the AFM contact mode, the cantilever is scanned across the sample at a fixed deflection. This allows one to determine the height of the samples (Z), and this represents an advantage over the other electronic microscope techniques. The AFM software allows the generation of a 3D image scan by the interaction between the tip and sample surface, and the tip deflection is correlated to the height of the sample through a laser and a detector.
In static mode (contact mode) with constant force, the output presents two different images: the height (z topography) and the deflection or error signal. Static mode is a valuable, simple imaging mode, especially for robust samples in air that can handle the high loads and torsional forces exerted by static mode. The deflection or error mode is operated in constant force mode. However, the topography image is further enhanced by adding the deflection signal to the surface structure. In this mode, the deflection signal is also referred to as the error signal as the deflection is the feedback parameter; any features or morphology that appear in this channel are due to the &#34;error&#34; in the feedback loop or, rather, due to the feedback loop required to maintain a constant deflection setpoint.
AFM&#39;s unique design makes it compact - small enough to fit on a tabletop - while also having high enough resolution to resolve atomic steps. The AFM equipment has a lower cost than the equipment for other electronic microscopes, and the maintenance costs are minimal. The microscope does not require a lab with special conditions such as a clean room or an isolated space; it only needs a vibration-free desk. For AFM, the samples do not need to undergo elaborate preparation like for other techniques (gold cover, slimming); only a dry sample has to be attached to the sample holder.
We use AFM contact mode to observe bacterial morphologies and the effects of NPs. The population and cellular morphology of bacteria fixed on a support can be observed, as well as the cellular damage produced by nanoparticles on the bacterial species. The images obtained by AFM contact mode confirm that it is a powerful tool and is not limited by reagents and complicated procedures, making it a simple, fast, and economical method for bacterial characterization.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AFM EasyScan 2</td>
			<td>NanoSurf</td>
			<td>discontinued</td>
			<td>Measurement Media</td>
		</tr>
		<tr>
			<td>bacteriological loop</td>
			<td>No aplica</td>
			<td>not applicable</td>
			<td>instrument for bacterial inoculation</td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1</td>
			<td>ThermoFisher Scientific</td>
			<td>4337455</td>
			<td>Matrix installation kit</td>
		</tr>
		<tr>
			<td>Bioedit</td>
			<td>not applicable</td>
			<td>version 7.2.5</td>
			<td>Sequence alignment editor</td>
		</tr>
		<tr>
			<td>Cary 60 spectrometer</td>
			<td>Agilent Technologies</td>
			<td>not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>ceftriazone</td>
			<td>Merck</td>
			<td>not applicable</td>
			<td>antibiotic</td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>eppendorf</td>
			<td>not applicable</td>
			<td>to remove particles that interfere with AFM</td>
		</tr>
		<tr>
			<td>ContAI-G Silicon cantilever</td>
			<td>BudgetSensors</td>
			<td>ContAl-G-10</td>
			<td>Measurement Media</td>
		</tr>
		<tr>
			<td>eosin and methylene blue agar</td>
			<td>Merck</td>
			<td>not applicable</td>
			<td>bacterial culture medium</td>
		</tr>
		<tr>
			<td>Escherichia coli</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25922</td>
			<td>bacterial strain</td>
		</tr>
		<tr>
			<td>GoTaq Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td>M8295</td>
			<td>PCR of 16S rRNA gene</td>
		</tr>
		<tr>
			<td>microplate</td>
			<td>Thermo Scientific</td>
			<td>10558295</td>
			<td>for microdilution analysis</td>
		</tr>
		<tr>
			<td>M&#252;ller-Hinton broth</td>
			<td>Merck</td>
			<td>not applicable</td>
			<td>bacterial culture medium</td>
		</tr>
		<tr>
			<td>nutrient agar</td>
			<td>Merck</td>
			<td>not applicable</td>
			<td>bacterial culture medium</td>
		</tr>
		<tr>
			<td>nutritious broth</td>
			<td>Merck</td>
			<td>not applicable</td>
			<td>bacterial culture medium</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>growth of bacteria</td>
		</tr>
		<tr>
			<td>Pseudomonas hunanensis 9AP</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>isolated from the garlic bulb by CNRG</td>
		</tr>
		<tr>
			<td>Sanger sequencing</td>
			<td>Macrogen</td>
			<td>not applicable</td>
			<td>sequencing service</td>
		</tr>
		<tr>
			<td>ScienceDesk Anti-Vibration workstation</td>
			<td>ThorLabs</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>slides</td>
			<td>not applicable</td>
			<td>not applicable</td>
			<td>glass holder for bacterial sample analysis</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 25923</td>
			<td>bacterial strain</td>
		</tr>
		<tr>
			<td>Thermalcycler</td>
			<td>Applied Biosystems</td>
			<td>Veriti-4375786</td>
			<td>PCR amplification</td>
		</tr>
		<tr>
			<td>Trypticasein soy agar</td>
			<td>BD</td>
			<td>BA-256665</td>
			<td>growth media</td>
		</tr>
		<tr>
			<td>ultrasonicator</td>
			<td>Cole-Parmer Ultrasonic Processor, 220 VAC</td>
			<td>not applicable</td>
			<td>for mixing the nanoparticle dilutions</td>
		</tr>
	</tbody>
</table>

contact mode atomic force microscopy as a rapid technique for morphological observation and bacterial cell damage analysis

Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the sample preparation for observation. Atomic force microscopy (AFM) is a very useful characterization technique due to its high resolution in three dimensions and because of the absence of any requirement for vacuum and sample conductivity. AFM can image a wide variety of samples with different topographies and different types of materials. 
AFM provides high-resolution 3D topography information from the angstrom level to the micron scale. Unlike traditional microscopy, AFM uses a probe to generate an image of the surface topography of a sample. In this protocol, the use of this type of microscopy is suggested for the morphological and cell damage characterization of bacteria fixed on a support. Strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), and Pseudomonas hunanensis (isolated from garlic bulb samples) were used. In this work, bacterial cells were grown in specific culture media. To observe cell damage, Staphylococcus aureus and Escherichia coli were incubated with different concentrations of nanoparticles (NPs). 
A drop of bacterial suspension was fixed on a glass support, and images were taken with AFM at different scales. The images obtained showed the morphological characteristics of the bacteria. Further, employing AFM, it was possible to observe the damage to the cellular structure caused by the effect of NPs. Based on the images obtained, contact AFM can be used to characterize the morphology of bacterial cells fixed on a support. AFM is also a suitable tool for the investigation of the effects of NPs on bacteria. Compared to electron microscopy, AFM is an inexpensive and easy-to-use technique.

Images of the morphology and size of S. aureus and P. hunanensis strains, as well as the population organization of both strains, were taken by atomic force microscopy in contact mode.&#160;The S. aureus images showed that its population was distributed by zones with aggregates of cocci (Figure 1A). With an increase in scale, there was a greater appreciation of the population distribution and morphology of the cocci (Figure 1B). The mi...

Watch this Scientific Journal Video about Contact Mode Atomic Force Microscopy as a Rapid Technique for Morphological Observation and Bacterial Cell Damage Analysis at JoVE.com

Contact Mode Atomic Force Microscopy as a Rapid Technique for Morphological Observation and Bacterial Cell Damage Analysis

Here, we present the application of atomic force microscopy (AFM) as a simple and fast method for bacterial characterization and analyze details such as the bacterial size and shape, bacterial culture biofilms, and the activity of nanoparticles as bactericides.

Electron microscopy is one of the tools required to characterize cellular structures. However, the procedure is complicated and expensive due to the ...

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Research

JoVE Journal

Biology

1.2K Views.  Universidad de Guadalajara. Here, we present the application of atomic force microscopy (AFM) as a simple and fast method for bacterial characterization and analyze details such as the bacterial size and shape, bacterial culture biofilms, and the activity of nanoparticles as bactericides.

Video: Contact Mode Atomic Force Microscopy as a Rapid Technique for Morphological Observation and Bacterial Cell Damage Analysis

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Characterizing Mechanical Properties of Primary Cell Wall in Living Plant Organs Using Atomic Force Microscopy

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the plant. Many sophisticated techniques have been developed to measure these properties; however, atomic force microscopy (AFM) remains the most convenient for studying cell wall elasticity at the cellular level. One of the most important limitations of this technique has been that only superficial or isolated living cells can be studied. Here, the use of atomic force microscopy to investigate the mechanical properties of primary cell walls belonging to the internal tissues of a plant body is presented. This protocol describes measurements of the apparent Young&#39;s modulus of cell walls in roots, but the method can also be applied to other plant organs. The measurements are performed on vibratome-derived sections of plant material in a liquid cell, which allows (i) avoiding the use of plasmolyzing solutions or sample impregnation with wax or resin, (ii) making the experiments fast, and (iii) preventing dehydration of the sample. Both anticlinal and periclinal cell walls can be studied, depending on how the specimen was sectioned. Differences in the mechanical properties of different tissues can be investigated in a single section. The protocol describes the principles of study planning, issues with specimen preparation and measurements, as well as the method of selecting force-deformation curves to avoid the influence of topography on the obtained values of elastic modulus. The method is not limited by sample size but is sensitive to cell size (i.e., cells with a large lumen are difficult to examine).

Studies of cell wall biomechanics are essential for understanding plant growth and morphogenesis. The following protocol is proposed to investigate thin primary cell walls in the internal tissues of young plant organs using atomic force microscopy.

The mechanical properties of the primary cell walls determine the direction and rate of plant cell growth and, therefore, the future size and shape of the ...

Measurement of Liver Stiffness Using Atomic Force Microscopy Coupled with Polarization Microscopy

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell behavior such as cell function, differentiation, and motility. However, as these processes are not homogeneous throughout the whole organ, it has become increasingly important to understand changes in the mechanical properties of tissues on the cellular level.
To be able to monitor the stiffening of collagen-rich areas within the liver lobes, this paper presents a protocol for measuring liver tissue elastic moduli with high spatial precision by atomic force microscopy (AFM). AFM is a sensitive method with the potential to characterize local mechanical properties, calculated as Young&#39;s (also referred to as elastic) modulus. AFM coupled with polarization microscopy can be used to specifically locate the areas of fibrosis development based on the birefringence of collagen fibers in tissues. Using the presented protocol, we characterized the stiffness of collagen-rich areas from fibrotic mouse livers and corresponding areas in the livers of control mice.
A prominent increase in the stiffness of collagen-positive areas was observed with fibrosis development. The presented protocol allows for a highly reproducible method of AFM measurement, due to the use of mildly fixed liver tissue, that can be used to further the understanding of disease-initiated changes in local tissue mechanical properties and their effect on the fate of neighboring cells.

We present a protocol to measure the elastic moduli of collagen-rich areas in normal and diseased liver using atomic force microscopy. The simultaneous use of polarization microscopy provides high spatial precision for localizing collagen-rich areas in the liver sections.

Matrix stiffening has been recognized as one of the key drivers of the progression of liver fibrosis. It has profound effects on various aspects of cell ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...

Fluorescence Lifetime Macro Imager for Biomedical Applications

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ranging from solid-state, O2-sensitive coatings to live animal tissue samples stained with soluble O2-sensitive probes. In particular, the nanoparticle-based near-infrared probe NanO2-IR, which is excitable with a 625 nm light-emitting diode (LED) and emits at 760 nm, was used. The imaging system is based on the Timepix3 camera (Tpx3Cam) and the opto-mechanical adaptor, which also houses an image intensifier. O2 phosphorescence lifetime imaging microscopy (PLIM) is commonly required for various studies, but current platforms have limitations in their accuracy, general flexibility, and usability.
The system presented here is a fast and highly sensitive imager, which is built on an integrated optical sensor and readout chip module, Tpx3Cam. It is shown to produce high-intensity phosphorescence signals and stable lifetime values from surface-stained intestinal tissue samples or intraluminally stained fragments of the large intestine and allows the detailed mapping of tissue O2 levels in about 20 s or less. Initial experiments on the imaging of hypoxia in grafted tumors in unconscious animals are also presented. We also describe how the imager can be re-configured for use with O2-sensitive materials based on Pt-porphyrin dyes using a 390 nm LED for the excitation and a bandpass 650 nm filter for emission. Overall, the PLIM imager was found to produce accurate quantitative measurements of lifetime values for the probes used and respective two-dimensional maps of the O2 concentration. It is also useful for the metabolic imaging of ex vivo tissue models and live animals.

This paper describes the use of a new, fast optical imager for the macroscopic photoluminescence lifetime imaging of long decay emitting samples. The integration, image acquisition, and analysis procedures are described, along with the preparation and characterization of the sensor materials for the imaging and the application of the imager in studying biological samples.

This paper presents a new photoluminescence lifetime imager designed to map the molecular oxygen (O2) concentration in different phosphorescent samples ...